EP0105014B1 - Rétablissement de tissu chez les animaux - Google Patents

Rétablissement de tissu chez les animaux Download PDF

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EP0105014B1
EP0105014B1 EP83450022A EP83450022A EP0105014B1 EP 0105014 B1 EP0105014 B1 EP 0105014B1 EP 83450022 A EP83450022 A EP 83450022A EP 83450022 A EP83450022 A EP 83450022A EP 0105014 B1 EP0105014 B1 EP 0105014B1
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tgf
composition
egf
growth factors
acid
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EP0105014A3 (en
EP0105014A2 (fr
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Michael Benjamin Sporn
Anita Bauer Roberts
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US Department of Health and Human Services
US Department of Commerce
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US Department of Commerce
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]

Definitions

  • This invention relates to a purified ⁇ -type transforming growth factor in the form of a homogeneous protein and to a method for its production as well as to compositions comprising this purified ⁇ -type transforming growth factor, and to the use of such compositions for producing a medicine.
  • EGF epidermal growth factor
  • TGF's transforming growth factors
  • NGF nerve growth factor
  • TGF transforming growth factor
  • TGFs were first discovered in the conditioned medium of virally-transformed neoplastic mouse cells, the application of the acid/ethanol method for extraction of peptides from tissues has now shown that TGFs can be found in almost all tissues, both neoplastic and non-neoplastic, from all species of animals that have been examined thus far.
  • TGF activity is usually measured with an in vitro phenotypic transformation assay, this does not imply that TGF activity in vivo is necessarily related to the development of malignancy.
  • the transformed phenotype is a physiological state associated with normal embryological development, and transforming ( onc ) genes have been found in normal cells of essentially all vertebrates. The function of these onc genes from normal cells is not known at present.
  • TGFs While there may be irreversible and excessive expression of TGF activity during malignant cell growth, the data at hand indicate that TGFs have a more benign and perhaps essential role in the function of normal cells. At present, it is not known what the intrinsic physiological roles of TGFs are. In this respect, TGFs are like many other peptide hormones or hormone-like agents which have recently been discovered and isolated; this is particularly true for many peptides of the nervous system, for which a defined chemical structure may be known, yet whose physiology is still a matter of uncertainty.
  • SGF sarcoma growth factor
  • TGFs have been described from diverse sources. These TGFs can be categorized into two groups: extracellular TGFs isolated from conditioned media of cultured cells, and intracellular (cell-associated) TGFs isolated by direct extraction of cells or tissues. Although extracellular TGFs have recently been isolated from non-neoplastic murine cells, use of conditioned medium has, in general, been restricted to neoplastic cell lines that could be grown in long-term, large-scale culture, including certain virally and chemically-transformed rodent cells and human tumor cell lines. The adaptation of an acid/ethanol extraction procedure to TGF isolation removed all limitations on cell types and quantities of tissues that could be examined.
  • extracts of all tissues or cells whether of neoplastic or non-neoplastic origin, whether from adult or embryonic tissue, whether from human, bovine, or murine genomes, have been shown to promote colony formation in a soft agar assay; hence, by definition, these extracts contain TGF activity.
  • the most commonly used indicator cell line is the rat kidney fibroblast cell clone, NRK 49F, which has been selected for its strong colony-forming response to the TGFs.
  • Rat-1 cells and mouse AKR-2B cells have also been used successfully as indicator cells.
  • TGFs referred to above are low molecular weight polypeptides which share with SGF the physical properties of acid and heat stability and sensitivity to treatment with both trypsin and dithiothreitol.
  • SGF the physical properties of acid and heat stability and sensitivity to treatment with both trypsin and dithiothreitol.
  • TGFs have some structural homology to EGF, since they compete with EGF for receptor binding.
  • Other TGFs do not compete with EGF for receptor binding, but instead are dependent on EGF for activity in the soft agar assay for colony formation.
  • TGF TGF-like growth factor
  • TGF- ⁇ are those TGFs which compete with EGF for receptor binding and which do not require EGF for the induction of colony formation in soft agar.
  • TGFs with these properties include SGF and other TGFs derived from neoplastic cells, as well as some TGFs from mouse embryos.
  • TGF- ⁇ are those TGFs which do not compete with EGF for receptor binding and which require EGF for the induction of colony growth in soft agar.
  • TGF- ⁇ represents the major colony-forming activity of the intracellular TGFs of both neoplastic and non-neoplastic cell lines and tissues. It can be assumed that TGF- ⁇ will be found in conditioned media as well, once the proper assays are used.
  • TGF- ⁇ gamma-type TGF
  • TGF- ⁇ delta-type TGF
  • TGF- ⁇ beta-type transforming growth factors
  • the subject-matter of the present invention is a purified ⁇ -type transforming growth factor in the form of a homogeneous protein, susceptible to Edman-degradative amino-acid sequencing which is obtainable by the steps of extracting murine, human or bovine cells or tissues with acid-ethanol to produce a cell extract, dialysing the cell extract to produce a dialysed acid-ethanol extract, applying the dialysed acid-ethanol extract to a gel-filtration chromotography column and eluting with 1M acetic acid to obtain a gel filtration eluate, adding the gel-filtration eluate to a reverse-phase HPLC C18 column using an acetonitrile gradient in 0.1% triflucroacetic acid to produce a C18 eluate, applying the C18 eluate to a CN reverse-phase HPLC column using a n-propanol gradient in 0.1% trifluoroacetic acid to obtain a CN eluate, and recovering from the CN-eluate a sequencible TGF
  • the present invention furthermore concerns a process for preparing the said purified beta-type transforming growth factor, comprising the above described process steps.
  • This invention further provides a composition for the promotion of cell proliferation and tissue repair whose active ingredient consists essentially of the above defined ⁇ -type transforming growth factor (TGF- ⁇ ) in amounts sufficient to promote cell proliferation.
  • TGF- ⁇ ⁇ -type transforming growth factor
  • composition further comprises as an additional ingredient at least one activating agent present in an amount sufficient to activate said TGF- ⁇ , wherein the activating agent is selected from the group consisting of epidermal growth factor (EGF) and ⁇ -type transforming growth factor (TGF- ⁇ ). Active ingredients are present in an amount sufficient to promote cell proliferation.
  • EGF epidermal growth factor
  • TGF- ⁇ ⁇ -type transforming growth factor
  • the present invention furthermore comprises the use of the above stated composition for the production of a medicine having a utility in the treatment of mammalian systemic traumata by systemic administration.
  • the TGF- ⁇ and the activating agent are preferably present in about equimolar amounts, and the active ingredients are present in an amount at least sufficient to stimulate cell proliferation (tissue repair).
  • the activated TGF- ⁇ compositions of this invention may be admixed with other (secondary) growth factors to enhance their activity.
  • compositions may be formulated in any suitable carrier for topical application, such as physiological saline solution or purified collagen suspension.
  • suitable carrier for systemic administration such as physiological saline solution or purified collagen suspension.
  • compositions of this invention are by direct application to a burn, wound, or other traumata situs. Periodic or continual further administration may be preferably indicated in most instances, since the active ingredients are physiologically utilized by the cells whose growth is being stimulated.
  • compositions of this invention may be administered systemically by injection, enterally, transdermal patches, and the like, depending upon the nature and site of the traumata to be treated.
  • the volume is adjusted with distilled water 60ml/10g of tissue and the tissue is minced. After overnight extraction at 4°C the mixture is centrifuged and the residue is reextracted for 2hrs with 40ml of a solution of 375ml of 95% ethanol, 105ml of distilled water and 7.5ml of concentrated HCl.
  • the combined supernatants are kept in the acidic range by adjustment to pH 5.2 with concentrated ammonium hydroxide followed by the addition of 1ml of 2 M ammonium acetate buffer, pH 5.3 per 85ml of extract. Two volumes of cold anhydrous ethanol and four volumes of cold anhydrous ether are immediately added, whereafter the mixture is allowed to stand at -20°C for 30-48 hours.
  • the resulting precipitate is collected by centrifugation or by rapid filtration through Whatman no. 1 paper and redissolved in 1 M acetic acid (3-4ml per gram of tissue).
  • the acetic acid-insoluble residue (10-25% of the total solvent-precipitated protein) which has little or no activity in the soft agar assay is discarded.
  • the samples After extensive dialysis at 4°C against 0.17 M acetic acid (Spectropor tubing, molecular weight cutoff 3500, Spectrum Medical Industries ) the samples are lyophilized to dryness.
  • the obtained samples may be redissolved in 1 M acetic acid before being subjected to chromatography. After that chromatography and high pressure liquid chromatography (HPLC) have been employed for further purification.
  • HPLC high pressure liquid chromatography
  • TGF activity measured by the ability to induce non-neoplastic indicator cells (NRK) to form colonies in soft agar, has been quantitated on an image analysis system with respect to both the number and size of the colonies formed.
  • NNK non-neoplastic indicator cells
  • TGF- ⁇ which elutes from the column earlier than marker EGF, is characterized by its ability to induce the formation of small colonies (850-3,100 ⁇ m2) in soft agar in the absence of added EGF and its ability to compete with EGF in a radio-receptor assay.
  • TGF- ⁇ which elutes later than TGF- ⁇ or marker EGF, does not compete with EGF for receptor binding and requires EGF to induce the formation of large colonies (>3,100 ⁇ m2) in the soft agar assay.
  • TGF- ⁇ from MSV-transformed 3T3 cells resembles SGF isolated from the conditioned medium of the same cells and other TGFs isolated from rat and human tumor cell lines.
  • SGF and the TGF- ⁇ 's from the conditioned media of a human melanoma cell line and virally-transformed rat embryo fibroblast have been purified to homogeneity.
  • the human melanoma TGF- ⁇ is a single chain polypeptide of molecular weight 7,400. Its amino acid composition and chromatographic behavior are markedly different from that of human EGF, but similar to that of murine SGF and rat TGF- ⁇ , suggesting that TGF- ⁇ 's from human, rat and mouse genomes are more closely related to each other than to EGF. There is therefore a reasonable possibility that TGF- ⁇ 's may have cross-species utility.
  • TGF- ⁇ In sarcoma virus-transformed rodent cell lines, the release of TGF- ⁇ into the medium has been correlated with the expression of the transformed phenotype, and within a selected set of human tumor cell lines that release TGF- ⁇ , the ability of the tumor cells to grow in soft agar has been correlated with the quantity of TGF- ⁇ they secrete.
  • secretion of TGF- ⁇ is not an absolute requirement for neoplastic behavior; certain chemically transformed murine cell lines and human lung carcinoma cell lines that do not secrete TGF- ⁇ release strong TGF activity that does not compete with EGF for receptor binding.
  • TGF- ⁇ of the acid/ethanol extract of the MSV-transformed 3T3 cells resembles other TGFs isolated from many neoplastic and non-neoplastic tissues.
  • TGF- ⁇ of the MSV-transformed cells eluted at the same position in the n -propanol gradient (48%) as one peak of TGF- ⁇ activity of the bovine salivary gland, and each was associated with a small peak of absorbance at 280 nm.
  • TGF- ⁇ 's one from a neoplastic mouse cell line and the other from a non-neoplastic bovine tissue, each migrated as a 12,500-13,000 daltons MW protein on SDS-PAGE in the presence of mercaptoethanol and as an apparent 25,000-26,000 daltons protein in the absence of mercaptoethanol; they therefore appear to be closely related to each other and different from both TGF- ⁇ and EGF.
  • the finding of TGF- ⁇ in all non-neoplastic tissues examined thus far suggests a normal physiological function for these TGFs. There is therefore a reasonable possibility that TGF- ⁇ 's may have cross-species utility.
  • TGF- ⁇ from bovine kidneys was purified 200,000-fold to the point of homogeniety.
  • TGF- ⁇ For amino-terminal sequence analysis, approximately 500 picomoles (M r 25,000) of TGF- ⁇ were reduced and S-carboxymethylated with dithiothreitol and iodo-[14C] acetic acid in the presence of 6M guanidine-HCl in 1 M Tris-HCl buffer, pH 8.4. Excess reagents were separated from carboxymethylated protein by HPLC on a 5 micron 50 x 4.6 mm column eluted with a gradient of 0-90% acetonitrile (1% per min) in 0.1% TFA. Overall recovery of the procedure was 96%, based on estimating the amount of protein by amino acid analysis using fluorescamine detection.
  • CMC S-carboxymethylcysteine
  • TGF- ⁇ TGF- ⁇
  • EGF EGF
  • EGF or TGF- ⁇ assayed by themselves, induced a maximal response of only a small number of colonies; this response was increased 10-fold by the addition of TGF- ⁇ .
  • the relative abilities of EGF and TGF- ⁇ to promote TGF- ⁇ -dependent formation of large colonies in soft agar correlated with their relative abilities to compete for binding to the EGF receptor; other experiments using chemically-modified EGF analogues have substantiated this correlation.
  • TGF- ⁇ and TGF- ⁇ suggest that these two TGFs may act through different pathways.
  • Experiments using TGFs of conditioned media of sarcoma virus-transformed rodent cells have shown that the synthesis of new RNA and protein is required before transformation occurs.
  • Other experiments have been directed at a possible role of TGFs in phosphorylation reactions.
  • Certain viral transforming gene products and their normal cellular homologues have tyrosine-specific protein kinase activity, and it has been proposed that phosphorylation at tyrosine of specific substrates may be important in the transformation process.
  • TGF- ⁇ Treatment of human carcinoma A431 cells with various TGFs derived from conditioned media of virally-transformed cells or human tumor cell lines (TGF- ⁇ ) resulted in phosphorylation of tyrosine residues in the 160 K EGF receptor.
  • the pattern of phosphorylation was indistinguishable from that induced by EGF itself, and thus would not appear to be transformation-specific.
  • dissolution of actin fibers of Rat-1 cells occurs when they are treated with either TGF or EGF. It is clear that further research is needed to establish the relationships of the TGFs to the retrovirus transforming gene products and the mode of action of the TGFs in neoplastic transformation.
  • TGF- ⁇ Synergistic interaction (activation) of TGF- ⁇ with TGF- ⁇ to induce the formation of large colonies of NRK cells in soft agar .
  • Soft agar colony-forming activity of varying concentrations of ⁇ Bondapak CN-purified TGF- ⁇ derived from MSV-transformed 3T3 cells was assayed either alone or in the presence of either CN-purified TGF- ⁇ derived from the same cells or murine EGF.
  • Soft agar colony-forming activity of varying concentrations of EGF or TGF- ⁇ was assayed either alone or in the presence of TGF- ⁇ .
  • TGFs were isolated on a relatively large scale from bovine sources and the wound healing activity of the compositions according to this invention were satisfactorily demonstrated using an experimental rodent wound healing protocol.
  • compositions according to this invention are effective in vivo , but also that TGFs may be employed cross-species.
  • TGF- ⁇ and TGF- ⁇ Bovine tissues, obtained fresh from the slaughterhouse and frozen immediately on dry ice, were extracted in 2kg batches with acid/ethanol in accordance with the above described process of A. B. Roberts, et al., Proc. Natl. Acad. Sci., U.S.A. , 77 :3494 (1980). Extracts from 6-8 kg tissue were combined and chromatographed on Bio-Gel P-30 with 1 M acetic acid, using an 80 liter bed volume column.
  • TGFs of extracts of bovine kidney or bovine salivary gland eluted in a broad peak between the RNase (13,700) and insulin (5,700) markers, as had been observed for the TGFs of mouse kidney and mouse salivary gland.
  • TGFs at this stage of purification had a specific activity approximately 10 to 25-fold higher than the acid/ethanol extracts, with a range of recovery of 150,000-200,000 colony-forming units per kg tissue.
  • Most of the in vivo studies reported below were done with salivary gland or kidney TGFS purified to this stage.
  • the TGFs activity in vitro was enhanced approximately 20-fold by the presence of 2-5 ng EGF per ml in the asssay, in accordance with this invention.
  • the bovine TGF- ⁇ were purified further by High Pressure Liquid Chromatography (HPLC) on ⁇ Bondapak C18 columns using an acetonitrile gradient in 0.1 percent trifluoroacetic acid, followed by a second HPLC step on ⁇ Bondapak CN columns using a gradient of n -propanol in 0.1 percent trifluoroacetic acid.
  • HPLC High Pressure Liquid Chromatography
  • each of the bovine TGF- ⁇ 's had an absolute requirement for EGF for colony-forming activity.
  • the yield of HPLC-purified TGF- ⁇ was approximately 20-100 ⁇ g per kg tissue, with a total activity of 7,000-18,000 colony-forming units.
  • the rats respond to these chambers as if they were wounds, and eventually the chambers become filled with fibroblasts and collagen.
  • the chambers become encapsulated with connective tissue, but there are few cells within the chambers themselves. There is thus a defined, enclosed space within the chambers, where a wound healing response can be quantitatively measured.
  • daily injections of TGF- ⁇ (0.1 ml, in sterile phosphate-buffered saline)into chambers A, B, and C were begun.
  • TGF- ⁇ 0.1 ml, in sterile phosphate-buffered saline
  • Chambers D, E, and F were used as controls, and were injected with either an amount of bovine serum albumin (BSA) alone or in combination with either TGF- ⁇ or EGF, such that the total protein was equivalent to the amount of TGF- ⁇ injected into chambers A, B, and C. Injections were made once daily for either 5 days (Table 2) or 9 days (Table 3). All injected materials were sterile. The rats were sacrificed 6 hours after the last TGF- ⁇ injection; in Table 3 they were injected with 0.5 mCi of thymidine-3H, specific activity 6.7 Ci/millimole (i.p.) together with the last TGF- ⁇ injection. The chambers were removed from the rats, all connective tissue on the outside of the wire mesh was peeled away, and then the contents of each chamber were determined.
  • BSA bovine serum albumin
  • Table 3 shows that 5 days of treatment of rats with TGF- ⁇ from either bovine salivary gland or bovine kidney caused a significant increase in total protein in the treated chambers, as compared to control chambers treated with an equivalent amount of bovine serum albumin (Experiments 1, 3).
  • the salivary gland TGF- ⁇ was still highly active after two steps of purification by the high pressure liquid chromatography (Experiment 2).
  • the effects observed are not the sole result of the minute amounts of EGF which had been used to potentiate the activity of TGF- ⁇ , since a highly significant difference between treated chambers A, B and C, compared to control chambers D, E and F was still observed when EGF was used as the control substance (Experiment 4).
  • compositions of this invention whose active ingredients are TGF- ⁇ activated by at least one of a TGF- ⁇ and an EGF, can reasonably be expected to have clinical use in the treatment of animals, particularly mammals, most particularly human beings. There are several sound bases for this conclusion.
  • compositions can markedly increase the growth of cells without changing their genotype.
  • An important characteristic of the components of the compositions of this invention is that they do not appear to be species specific. That is, TGF- ⁇ from one species can be activated by TGF- ⁇ and/or EGF from other species.
  • the cells whose growth is promoted can be of any type such as fibroblast or epithelial, although it is considered that the growth promotion of fibroblast cells will have the greatest medical utility.
  • compositions of this invention have utility in the treatment of traumata by the rapid promotion of the proliferation of the cells surrounding the traumata.
  • compositions of this invention Two types of application of the compositions of this invention are contemplated.
  • the first, and preferred, application is topically for the promotion of surface wound healing.
  • wound or other traumata that can be treated, and these include (but are not limited to): first, second and third degree burns (especially second and third degree); surgical incisions, including those of cosmetic surgery; wounds, including lacerations, incisions, and penetrations; and surface ulcers including decubital (bed-sores), diabetic, dental, haemophiliac, and varicose.
  • first, second and third degree burns especially second and third degree
  • surgical incisions including those of cosmetic surgery
  • wounds including lacerations, incisions, and penetrations
  • surface ulcers including decubital (bed-sores), diabetic, dental, haemophiliac, and varicose.
  • the compositions may also be useful for minor wounds, and for cosmetic regeneration of cells such as epithelial. It is also contemplated that the compositions may be utilized by the topical application to internal surgical incisions.
  • compositions When applied topically, the compositions may be combined with other ingredients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, and cannot degrade the activity of the active ingredients of the compositions.
  • the compositions of this invention When the compositions of this invention are applied to burns, they may be in the form of an irrigant, preferably in combination with physiological saline solution.
  • the compositions can also be in the form of ointments or suspensions, preferably in combination with purified collagen.
  • the compositions also may be impregnated into transdermal patches, plasters, and bandages, preferably in a liquid or semi-liquid form.
  • the second application is systemically for the healing of internal wounds and similar traumata. Such an application is useful provided that there are no, or limited, undesirable side-effects, such as the stimulation of neoplastic cellular growth.
  • compositions When applied systemically, the compositions may be formulated as liquids, pills, tablets, lozonges, or the like, for enteral administration, or in liquid form for parenteral injection.
  • the active ingredients may be combined with other ingredients such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, and cannot degrade the activity of the active ingredients of the compositions.
  • TGF- ⁇ s or EGFs The amount of activating agent (TGF- ⁇ s or EGFs) present depends directly upon the amount of TGF- ⁇ s present in the activated compositions of this invention. There are indications that the activation is not catalytic in nature, and that therefore approximately stoichiometric (equimolar) quantities are preferred.
  • the amount of activated composition to be used in the methods of this invention cannot be stated because of the nature of the activity of TGFs and the nature of healing wounds and/or other traumata.
  • the TGFs activate cells by binding to receptor sites on the cells, after which the TGFs are absorbed and utilized by the cells for the synthesis of new protein, resulting in cell multiplication.
  • the TGFs are consumed by the cell regenerating process itself, rather than acting in an enzymatic or other catalytic manner.
  • Receptors for EGFs have been found on a wide variety of fibroblastic, epithelial, and parietal cells, as disclosed in Gonzalez, et al., J. Cell. Biol. , 88 :108-144 (1981).
  • EGF binding (receptor) sites for each rat intestinal epithelial cell, as disclosed in M. E. Lafitte, et al., FEBS Lett. , 114(2) :243-246 (1980). It must also be obvious that the amount of a cell growth promoting substance (such as the compositions of this invention) that must be utilized will vary with the size of the wound or other traumata to be treated.
  • compositions of this invention both provoke and sustain cellular regeneration, a continual application or periodic reapplication of the compositions in indicated.
  • the amount of active ingredient per unit volume of combined medication for administration is also very difficult to specify, because it depends upon the amount of active ingredients that are afforded directly to the regenerating cells of the wound or other traumata situs.
  • the TGF- ⁇ s should preferably be present in an amount of at least about 1.0 nanogram per milliliter of combined composition, more preferably in an amount up to about 1.0 milligram per milliliter.
  • the activated transforming growth factors of this invention may be physically admixed with one or more of many other (secondary) peptide and non-peptide growth factors. Such admixtures may be administered in the same manner and for the same purposes as the activated transforming growth factors of this invention utilized alone, to enhance their activity in promoting cell proliferation and repair.
  • the useful proportions of activated transforming growth factor to secondary growth factors are 1:.1-10 mols, with about equimolar amounts being preferred.
  • the secondary growth factors may be used alone or in any physiologically and pharmaceutically compatible combination.
  • the known secondary growth factors in approximately descending order of usefulness in this invention (by group), include:
  • This invention also incorporates the inactive intermediate substance TGF- ⁇ per se .
  • TGF- ⁇ Prior to this invention, this substance had not been isolated or identified.
  • TGF- ⁇ is believed to be substantially the same or very similar for each animal species, regardless of the individual of that species or the particular body cells from which it is derived. Since TGF- ⁇ has been shown to be non-species-specific between rodents, cattle, and human beings, it is also reasonable to believe that the substance is substantially the same or very similar when derived from any mammal, and possibly from any animal source. It should be noted, moreover, that this invention includes TGF- ⁇ regardless of the source from which it is isolated or derived, including genetically engineered cells. It is well within the capabilities of biochemical technology to genetically engineer a cell to produce TGF- ⁇ at the present time.
  • TGF- ⁇ has no wound-healing or other tissue-repair activity unless it has been activated by an agent as described above.
  • TGF- ⁇ per se may be administered, in accordance with this invention, instead of activated TGF- ⁇ , when there are sufficient endogenous activating agents present in an animal, to activate an amount of TGF- ⁇ sufficient to promote cell proliferation and tissue repair. It is anticipated that in an animal suffering from the traumata contemplated herein, there usually will not be sufficient endogenous activating agents present.

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Claims (19)

  1. Facteur de croissance transformant de type β purifié ayant la forme d'une protéine homogène, sensible à un séquençage en acides aminés par la dégradation de Edman, et qui peut être obtenu par les étapes de :
    a) extraction de cellules ou tissus murins, humains ou bovins par l'acide-éthanol pour produire un extrait cellulaire ;
    b) dialyse de l'extrait cellulaire pour produire un extrait à l'acide-éthanol dialysé ;
    c) application de l'extrait à l'acide-éthanol dialysé sur une colonne de chromatographie par filtration sur gel et élution avec de l'acide acétique 1M pour obtenir un éluat de filtration sur gel ;
    d) addition de l'éluat de filtration sur gel sur une colonne C₁₈ de HPLC en phases inversées utilisant un gradient d'acétonitrile dans de l'acide trifluoroacétique à 0,1 % pour produire un éluat C₁₈ ;
    e) application de l'éluat C₁₈ sur une colonne de HPLC en phase inversée CN utilisant un gradient de n-propanol dans de l'acide trifluoroacétique à 0,1 % pour obtenir un éluat CN ; et
    f) récupération dans l'éluat CN d'un TGF-β séquençable qui migre comme une protéine de poids moléculaire compris entre 12 500 et 13 000 daltons sur un gel de SDS-PAGE réducteur et comme une protéine de poids moléculaire compris entre 25 000 et 26 000 daltons sur un gel de SDS-PAGE non réducteur.
  2. Facteur de croissance selon la revendication 1, provenant d'une cellule modifiée par génie génétique.
  3. Facteur de croissance selon la revendication 1, provenant de rein de boeuf.
  4. Facteur de croissance transformant selon l'une des revendications 1 à 3, possédant une séquence partielle d'acides aminés déterminée par la dégradation de Edman, constituée des éléments suivants pour chacune de ses deux sous-unités :
    Figure imgb0008
    CMC étant de la semi-cystine ou cystéine déterminée en tant que S-carboxyméthylcystéine.
  5. Procédé de préparation du TGF-β séquençable purifié, provenant d'une cellule ou d'un tissu murin, humain ou bovin, comprenant les étapes de la revendication 1.
  6. Composition destinée à la stimulation de la prolifération cellulaire et de la réparation tissulaire chez l'animal et l'homme, dont le principe actif est constitué essentiellement par un facteur de croissance transformant de type β (TGF-β) selon l'une quelconque des revendications 1 à 4 en des quantités suffisantes pour stimuler la prolifération cellulaire.
  7. Composition selon la revendication 6, dans laquelle ledit TGF-β est présent une quantité comprise entre 1,0 nanogramme et 1 milligramme par ml de composition.
  8. Composition selon les revendications 6 ou 7, contenant en outre au moins un véhicule pharmaceutiquement acceptable.
  9. Composition selon la revendication 8, dans laquelle le véhicule est un collagène purifié et la composition est une suspension destinée à l'application topique.
  10. Composition selon la revendication 8, dans laquelle le véhicule est un sérum physiologique et la composition est un irrigant.
  11. Composition selon l'une quelconque des revendications 6 à 10, comprenant en outre comme principe actif au moins un agent activant présent en une quantité suffisante pour activer ledit TGF-β, l'agent activant étant sélectionné dans le groupe constitué par des facteurs de croissance épidermiques (EGF) et des facteurs de croissance transformants de type α (TGF-α), lesquels principes actifs sont présents en une quantité suffisante pour stimuler la prolifération cellulaire.
  12. Composition selon la revendication 11, utilisée pour la stimulation topique de la prolifération de cellules fibroblastiques chez le mammifère, dans laquelle le TGF-β et au moins un agent activant sont présents en des quantités approximativement équimolaires.
  13. Composition selon la revendication 12, dans laquelle l'agent activant est un EGF ou TGF-α.
  14. Composition selon la revendication 11, dans laquelle le TGF-β activé est mélangé avec au moins un facteur de croissance secondaire dans un rapport molaire de 1:0,1-10, de préférence en des quantités approximativement équimolaires.
  15. Composition selon la revendication 11, dans laquelle le facteur de croissance secondaire est au moins un des éléments du groupe constitué par : des facteurs de croissance d'origine plaquettaire, des facteurs de croissance fibroblastiques, des facteurs de croissance angiogéniques, des facteurs de croissance de type insuline, l'insuline, des facteurs de croissance nerveux et des stéroïdes anabolisants.
  16. Emploi d'une composition selon l'une quelconque des revendications 6 à 15 pour la préparation d'un médicament destiné au traitement de lésions, brûlures, ulcères et autres traumatismes topiques mammaliens par application topique.
  17. Emploi de la composition selon la revendication 16, dans lequel le médicament est destiné à une réadministration topique périodique ou continue.
  18. Emploi d'une composition selon l'une quelconque des revendications 6 à 15 pour la préparation d'un médicament utile dans le traitement des traumatismes systémiques mammaliens par administration systémique.
  19. Emploi d'une composition selon la revendication 18, dans lequel le médicament est destiné à une réadministration systémique périodique ou continue.
EP83450022A 1982-09-24 1983-09-22 Rétablissement de tissu chez les animaux Expired - Lifetime EP0105014B1 (fr)

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